This project is designed to define the morphologic, molecular, and metabolic characteristics of breast ducts and ductal epithelial cells at normal risk and at high risk for breast cancer among Caucasian, Hispanic and African American women. This information is needed to define the early changes in the carcinogenic pathway for breast cancer, to develop an improved classification and molecular signature of preneoplastic breast tissue for risk assessment, to identify new targets and to facilitate selection and monitoring of women for breast cancer prevention, and to define the molecular basis for disparities in the development and presentation of breast cancer. This project includes the following clinical and laboratory studies: a.) Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and Gene Expression Profile (DN Danforth, PI). Protocol 02-C-0077 characterizes by ductal lavage and ductal endoscopy the breast ducts and ductal epithelium of Caucasian, African American, and Hispanic women at normal risk and at high risk for breast cancer. One hundred forty women have been studied, 66 high risk subjects and 74 subjects at normal risk. A significantly improved method of ductal epithelial cell sampling has been developed under this study which provides multiple samples of pure (90%) ductal epithelial cells with high cellularity. These important findings were recently published (Danforth, DN et al, Breast Cancer: Basic and Clinical Research, 9:31-40, 2015). Extraction of a single intact ductal lavage sample (fluid and epithelial cells) or the separate frozen cellular component provided DNA and RNA suitable for multiple downstream molecular studies including RT-PCR of miRNA species, qPCR of the telomerase gene, whole genome DNA amplification, arrayCGH analysis, and microarray gene expression profiling. This method significantly expands our ability to define the molecular characteristics according to breast cancer risk of breast ductal epithelium in women at different risks for breast cancer, and also introduces a much needed method for collecting ductal epithelial cells from women at normal risk for breast cancer, a critical control group for defining the multiple molecular characteristics of women at increased risk for breast cancer. It has been shown that 50% - 70% of women in the U.S. who develop breast cancer have no identifiable risk factors, and thus our findings should also permit us to further characterize genomic changes and identify at-risk women in this group, a major population of women in the U.S. To compliment and guide the molecular studies in this protocol, a comprehensive literature review was conducted to identify and define genomic changes in normal breast tissue at normal risk and at high risk for breast cancer (Danforth, DN. Breast Cancer: Basic and Clinical Research 10:109, 2016). This indicated that normal risk breast tissues (such as reduction mammoplasty) contain evidence of early breast carcinogenesis including loss of heterozygosity, DNA methylation of tumor suppressor and other genes, and telomere shortening. In normal tissues at high risk for breast cancer (such as normal breast tissue adjacent to breast cancer or the contralateral breast), these changes persist, and are increased and accompanied by aneuploidy, increased genomic instability, a wide range of gene expression differences, development of large cancerized fields, and increased proliferation. A model for the carcinogenic pathway in normal risk and in high risk normal breast tissue is proposed in this publication. Cytologic studies of ductal cells under protocol 02-C-0077 has revealed the presence of atypical epithelial cells in 26.5% of normal risk and 22.9 % of high risk subjects. These subjects were further evaluated by ductal endoscopy and breast MRI, which did not indicate an associated atypical hyperplasia lesion in any subject. Gene expression profiling studies did not demonstrate any gene expression differences in subjects with atypia vs. those without atypia. On follow-up, 2 high risk subjects and no normal risk subjects developed invasive breast cancer. Together these findings suggest epithelial atypia on ductal lavage may be of prognostic significance in selected women at high risk, but not in normal risk subjects. Gene expression studies did, however, demonstrate downregulation of multiple expressed genes among high risk vs. normal risk subjects. Of particular interest, these gene expression changes were predominantly associated with intraepithelial lymphocytes, suggesting that loss of immune surveillance may be an important characteristic associated with a high risk for breast cancer, and might potentially contribute to progression of genomic changes in these women. These findings also indicate that the ductal sampling technique described above collects the entire cellular content of the ducts, including the terminal lobular units, which allows for a comprehensive analysis of breast ductal cells including both intraepithelial lymphocytes and epithelial cells. Breast ductal fluid and ductal epithelial cells are being studied for the presence of miRNA, noncoding transcripts which bind to mRNA and result in gene silencing. miRNA has been identified in exosomes of breast ductal fluid collected from these subjects, suggesting an important mechanism for the expansion of the cancerized field and area at risk within the breast and enhancement of progression through the carcinogenic pathway. We are also developing in this project a potentially very valuable resource, the development of normal breast epithelial cell lines from the ductal lavage samples from women at different risks for breast cancer. We are developing these cell lines in conjunction with Lonza Corp (Walkersville, MD). Demographic data including detailed risk assessment information is available for each subject. The development of a panel of cell lines according to different risks for breast cancer will provide abundant material for molecular profiling of risk characteristics, facilitate analysis of metabolic properties including response to exogenous mitogens and inhibitory substances, promote identification of alterations in signaling pathways according to risk, and allow development of an in vitro model useful for evaluation of chemoprevention drugs. Lastly, to compliment our studies of the basis of racial studies, a comprehensive review of the literature has recently been published proposing for the first time a model describing the relationship of biological and nonbiological factors to the initiation and development of the major disparities in breast cancer between African American and Caucasian women (Danforth, DN Breast Cancer Research, 15:208-220, 2013). This model identified multiple molecular differences in breast cancer between African American and Caucasian women, and these differences are the major drivers of the disparities in age of onset, more advanced stage, more aggressive histology, and worse survival in African American vs. Caucasian women. Multiple socioeconomic, reproductive and health care factors influence the outcome of the disparities through their influence on the breast cancer molecular characteristics. This model also emphasizes the need for defining the molecular characteristics of early carcinogenesis in these ethnic groups.